It is now well established that the wild-type p53 gene encodes a protein that has properties of a negative growth regulator which can apparently function as a tumor suppressor. Allelic deletions and mutations in the gene have been associated with a wide range of human tumors. Simple inactivation of p53s tumor suppressing activity is usually not sufficient for transformation and a number of studies indicate that mutant p53 may acquire novel activities and function as a transforming gene. In many transformed cells and primary tumors, mutant p53 proteins are detected at elevated levels. Both mutations in the gene and their overexpression appear to be required for its transforming capability. Although high level expression of p53 is a common feature of many transformed cells, much remains to be learned about the molecular basis for this increased gene expression. The major goal of our research presented in this grant application is to define the mechanisms that lead to de-regulated expression of the p53 gene. We will identify factors that are involved in regulating p53 gene expression and why mutant forms of the gene are expressed at high levels in transformed cells. Preliminary findings indicate that the proto- oncogene product c-Myc as well as the cellular transcription factor USF, through direct binding to the promoter, may play a role in regulating expression of the p53 gene. In order to extend these findings, a detailed analysis of the role of c-Myc and USF in expression of the p53 gene will be carried out. We will examine the binding of these factors to the p53 promoter in vitro as well as the functional effect that that binding has on the activity of the promoter by carrying out co-transfection assays where the p53 promoter will be controlling the expression of a reporter gene. Strategies will be developed, employing both inducible anti-sense and sense constructs, to alter the levels of c-Myc and USF in the cell to study their in vivo physiological effects on p53 expression. In addition, a thorough mutational analysis of the p53 regulatory regions will be carried out in order to define the transcriptional regulatory elements, both cis- and trans-, of the promoter and to examine the basis of enhanced expression of the gene in transformed cells. Finally, it will be determined whether naturally occurring mutations in regulatory elements of the p53 gene play a role in inactivating expression of the gene in myeloid tumors. To examine this possibility, segments of the p53 regulatory sequences from various tumors will be amplified by the polymerase chain reaction and analyzed for mutations and alterations in activity. These studies will provide information on the mechanisms leading to the de- regulated expression of p53 that is seen in a wide spectrum of transformed cells.